The invention is in the field of eukaryotic cell culture, and improved methods of recombinant protein production. More specifically, the invention relates to the modulation of the IGF-1 signaling pathway in cultured eukaryotic cells so as to obtain cell lines that can be used in serum-free and/or protein-free and/or peptone-free media.
Serum is often used for the propagation of mammalian cell lines. However, when mammalian cells are used for the production of recombinant proteins, there is increasing pressure to remove serum from the manufacturing process. Some of the driving reasons to implement serum-free cell-culture technology are the expense of serum, variation between serum lots and serum quality, regulatory concerns regarding biological agents in serum and the burden of removing serum proteins in downstream processing. (Adamson R., 1994, Ann. Hematol. 68 Suppl 3: S9-14; Thomas et al., Animal Cell Technology: Products of Today, Prospects for Tomorrow (Spier R E, Griffiths J B and Berthold W (ed.) pp. ESACT Butterworth-Heinemann, 1994)). There is also a recognized need in the art, for reasons of reduced cost and increased media consistency, for production cell lines able to grow in medium free of peptone additives. In addition, peptide growth factors are one of the most expensive media components; removal of such growth factors achieves significant cost reduction.
Adaptation of recombinant production cell lines to serum-free growth can be a time consuming step in process development with variable effects on recombinant protein expression and protein quality (Barnes et al., 1980, Anal. Biochem. 102: 255-270; Evans et al., 1956, Cancer Res. 16: 77-86; Hamilton et al., 1977, In Vitro 13: 537-547; Sinacore et al., 1996, Biotech. Bioeng. 52:518-528). For example, CHO cell lines have been adapted by Gandor et al. to growth in medium free not only of serum and growth factors (such as insulin) but of all proteins (Gandor et al. 1995, FEBS Lett. 377: 290-294). With this adaptation, however, came difficulties caused by cell line reversion. The cells, which were initially DHFR-negative, reverted to a DHFR-positive phenotype during prolonged continuous culture in the serum-free medium. Other investigators found that previously adapted serum-free cultures reverted to serum-dependent phenotype when cultured in serum-containing media (Yao et al., 1991, Proc Natl Acad Sci USA 88: 9422-9425).
Another cell line, termed xe2x80x9cVeggie-CHO,xe2x80x9d was adapted to be able to grow in serum-free and protein-free medium while still being DHFR deficient (Rasmussen et al., 1998, Cytotechnology 28:31-42). The adaptation process involved the gradual reduction of serum supplementation in the media and the replacement of serum with low levels of growth factors, IGF-1 and transferrin, in an enriched cell growth medium. The cells grown in serum-free medium were then weaned off these growth factors. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hours in continuous growth cultures over a period of three months and have retained the DHFR-deficient phenotype of their parental DXB 11-CHO cells (Id.). However, the process of achieving Veggie-CHO was time consuming and required over 160 passages. Additionally, there was an enormous difference in the cellular response (as measured by viability and doubling time) to growth in medium with low concentrations of serum and growth in medium with no serum. Only by making a gradual transition to medium without serum, with a prolonged adaptation period, were viable and stable (DHRFxe2x88x92) cell lines obtained.
One method that has been proposed to generate cell lines that can grow in media with reduced cytokines, hormones and growth factors is to cause the cells to express a bcl-2 gene (WO 93/20200 by Evan et al.). Evan et al. reported that if one took myeloma/hybridoma cells (which were chosen as cells that express essentially no bcl-2 MRNA or protein), and introduced into them a bcl-2 expression construct, the resulting cell lines had more stress resistance and decreased requirements for fetal calf serum (Id.). However, complete removal of serum and growth factors from the medium in which the hybridoma cells were grown was not reported (Id.). In another study that was directed to analyzing the signaling of the kinase MEK1, Greulich and Erickson transfected NIH-3T3 cells with an expression vector encoding for a fusion protein containing a constitutively activated MEK1 mutant (termed DD) fused to the hormone-binding domain of the estrogen receptor (encoding a product termed xe2x80x9cMek-ERxe2x80x9d) (Greulich et al., 1998, J. Biol. Chem. 273:13280-13288). Addition of the synthetic ligand 4-hydroxytamoxifen presumably activated Mek-ER. These authors reported that in the presence of 4-hydroxytamoxifen, the Mek-ER expressing cells could proliferate in low (0.5%) serum (Id.). However, when the cells were grown in medium without 4-hydroxytamoxifen but with 10% serum, they proliferated three times as fast. Thus, they concluded that expression of MEK1 could not override the requirement for serum and growth factors in these cells.
Yet another cell line adapted to growth in medium free of both serum and growth factors has been termed xe2x80x9cSuper CHOxe2x80x9d (Pak et al., 1996, Cytotechnology 22:139-146; see also WO 97/05240). In order to overcome the need for exogenously added growth factor, expression vectors containing the genes for transferrin and IGF-1 were transfected into CHO cells (Id.). In addition, EP 0 666 312 describes a method of generating cell cultures that can proliferate in serum-free and protein-free medium by transfecting cells with an expression vector that encodes the cell-cycle regulatory proteins cyclin E and/or transcription factor E2F-1.
Although many technical advances have been made, there still remains a need in the art to rapidly, and reliably, generate industrially important cell cultures that can proliferate in serum-free and/or protein-free and/or peptone-free media. The present invention is directed at fulfilling this need.
The invention is based, in part, on the discovery that cells that have been adapted over many generations to growth in serum-free and protein-free medium, Veggie-CHO cells, have alterations in the intracellular IGF-1 receptor signaling cascade. The present inventors found that the advantageous phenotypes of the Veggie-CHO cells could be duplicated in a more controlled, consistent and reliable manner by genetically engineering individual components of the IGF-1 signaling pathways.
Accordingly, in one aspect, the invention provides an eukaryotic host cell genetically engineered to express a gene for a protein of interest and at least one IGF-1-signaling pathway gene. Preferred IGF-1-signaling pathway genes are a PKB gene (e.g., PKBxcex1, PKBxcex2 and PKBxcex3), a MEK gene (e.g., MEK1 and MEK2), a glut5 gene, a glut 1 gene, an ERK gene (e.g., ERK1, also known as MAPK p44, and ERK2, also known as MAPK p42), a JNK gene, a 14-3-3 protein gene, a PDK gene, an IRS gene, and a PI3 kinase gene. The protein of interest can be any recombinant protein of economic interest. Examples of such proteins include but are not limited to a soluble TNF receptor, a soluble IL4 receptor, a soluble IL-1 type II receptor, a soluble Flt3 ligand, a soluble CD40 ligand, an erythropoeitin, an antibody, and hormones, to name just a few. Optionally, the gene for the protein of interest and/or the IGF-1-signaling pathway gene(s) can be linked to a selectable marker. Preferred host cells are mammalian cells, and more preferably mammalian cells that are grown in culture. In addition, the host cell can be adapted to grow in serum-free and/or protein-free and/or peptone-free medium.
In another related aspect, the invention provides a method of producing a protein of interest, the method comprising culturing an eukaryotic host cell genetically engineered to express a gene for a protein of interest and at least one IGF-1-signaling pathway gene under conditions such that the protein of interest is expressed. Optionally, the method entails collecting and/or purifying the protein of interest from the cell culture. Such methods are particularly advantageous for culturing the host cells in serum-free and/or growth-factor free and/or protein-free and/or peptone-free media. In addition, the methods of the invention frequently improved yields of the protein of interest.
In still another aspect, the invention relates to a method of producing a cell for production of a protein of interest, the method comprising genetically engineering a cell to express a gene that encodes a protein of interest, and to express an IGF-1-signaling pathway gene. The cells can be genetically engineered in any order or simultaneously.
Another aspect of the invention is a method of producing a mammalian cell line capable of growth in serum-free medium, the method comprising exposing cells that have been genetically engineered to overexpress or underexpress at least one IGF-1-signaling pathway gene to serum-free medium, and isolating a cell line that grows in serum-free medium. In alternative or additional embodiments, the cells are exposed to protein-free and/or peptone-free medium, and cell lines that grow in protein-free and/or peptone-free medium are isolated. Preferred IGF-1-signaling pathway genes are MEK genes, MAP kinase genes, and PKB genes.